Published on Web 09/06/2003
Coordination and Mechanism of Reversible Cleavage of S-Adenosylmethionine by the [4Fe-4S] Center in Lysine 2,3-Aminomutase Dawei Chen,† Charles Walsby,‡ Brian M. Hoffman,‡ and Perry A. Frey*,† Department of Biochemistry, UniVersity of Wisconsin-Madison, 1710 UniVersity AVenue, Madison, Wisconsin 53726, and Department of Chemistry, Northwestern UniVersity, 2145 Sheridan Road, EVanston, Illinois 60208 Received May 13, 2003; E-mail:
[email protected] Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of L-lysine and L-β-lysine, eq 1, by a radical mechanism initiated by the reversible, reductive homolytic scission of the C5′-S bond in S-adenosylmethionine (SAM) to form methionine and the 5′deoxyadenosyl radical at the active site.1-9 LAM is a member of a
superfamily of enzymes in which a [4Fe-4S]+ cluster with a unique, noncysteinyl coordinated Fe provides the electron required in the cleavage of SAM.11-21 Little is known of the mechanism by which the electron is inserted into SAM to effect its cleavage, and it is not known whether all enzymes of the family employ the same mechanism. Selenium X-ray absorption spectroscopy (XAS) of an intermediate in the LAM reaction where Se-adenosyl-L-selenomethionine (SeSAM) replaces SAM shows that electron transfer from the [4Fe-4S] center occurs by an inner sphere mechanism culminating in direct ligation of selenomethionine to iron upon cleavage of SeSAM.22 In this paper, we use ENDOR spectroscopic methods first employed in studies of the related enzyme, pyruvate formate lyase activating enzyme (PFL-AE),23,24 to show that SAM binds to LAM by chelating the unique iron of the [4Fe-4S] center through the carboxylato and amine groups of its L-methionine moiety. Through combination of the ENDOR and XAS spectroscopic results, we postulate a mechanism for the cleavage of SAM that involves multiple ligation with the [4Fe-4S] center. Experiments have been performed on samples with the Fe-S cluster reduced to the 1+ state with dithionite, denoted as [1+/SAM], and reduced samples trapped in the geometry of the 2+ state. The latter were prepared by freezing the enzyme with its cluster in the 2+ state, and then cryoreducing to generate the 1+ state within its frozen matrix; these are denoted as [2+/SAM]red. Spectra were obtained using SAM labeled with 17O in the carboxylato group, with 15N in the R-amino group, and with either 13C or 2H in the methyl group.25-27 The EPR spectra of both 3 [1+/SAM] and [2+/SAM]red are characterized by g ) [∼2.0, 1.90, 1.85] (see Supporting Information Figure S1).27,28 The ENDOR spectra of [1+/17O-SAM] in Figure 1 show a broad, asymmetric feature that is absent in the unlabeled sample. This is assigned to the ν+ branch of the 17O ENDOR response from the 17O-carboxylate of SAM. The large coupling (A(17O) ) 11.4 MHz at g2) is comparable to that found for the corresponding complex of PFLAE24 and arises from a carboxylato oxygen coordinated directly to the unique Fe of the cluster. The ENDOR spectra of unlabeled [1+/ SAM] show the ν+ branch of a 14N signal; it disappears in the spectrum of [1+/15N-SAM] and is replaced by a well-resolved † ‡
University of Wisconsin-Madison. Northwestern University.
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Figure 1. 35 GHz Davies pulsed 17O- and 15N-ENDOR spectra of LAM[4Fe-4S]+ in complex with 17O- and 15N-SAM.23b The upper spectrum is that of [1+/17O-SAM], and the lower is of [1+/15N-SAM]. The offsets displaying greater noise are of [2+/17O-SAM]red and [2+/15N-SAM]red. Conditions: MW pulse lengths 80, 40, 80 ns; Rf pulse, 60 µs; T ) 2 K.
ν+ peak from a 15N with a corresponding coupling A(15N) ) 9.1 MHz at g2 (Figure 1). Such a coupling requires that the amino group of SAM is coordinated to the unique Fe of the 1+ LAM cluster; the coupling indeed is substantially larger than that of 15N-SAM bound to the cluster of PFL-AE.24 Thus, as with PFLAE,24 the ENDOR spectra taken with [1+/SAM (14N, 17O)] disclose that the carboxylato O and amino N of the SAM methionine moiety form a five-membered-ring chelate of the unique iron of the LAM [4Fe-4S] cluster. However, the differences in 14,15N couplings indicate that details of this chelate structure must be different in the two enzymes. ENDOR spectra taken at g2 for [1+/SAM] prepared with SAM labeled at the methionine methyl group by 13C and 2H show signals from both labels (not shown). For 13C, the doublet splitting corresponds to A(13C) ) 0.7 MHz. The 2H (I ) 1) spectrum includes an unresolved quadrupole splitting of each branch; the total breadth of the pattern corresponds to A(2H) + 3P ) 0.7 MHz, taking as a probable value23 that 3P ≈ 0.1 MHz yields A ≈ 0.6 MHz. Preliminary 2-D field-frequency plots for the 13C ENDOR signal from [1+/13C-SAM] show a distinctly dipolar pattern, with the maximum coupling of A ) 0.8 MHz (not shown), which is larger than the maximum coupling observed from the analogous sample with PFL-AE, A(13C) ) 0.5 MHz.23 Unlike that enzyme, the preliminary 2-D field-frequency 13C ENDOR pattern suggests that there is little or no isotropic component to the coupling. This would 10.1021/ja036120z CCC: $25.00 © 2003 American Chemical Society
COMMUNICATIONS Scheme 1
References (1) (2) (3) (4) (5) (6) (7) (8)
indicate that the methyl group is closer to Fe in LAM, but with little or no covalent interaction between a negatively charged sulfide of the cluster and the positively charged sulfur of the SAM.23 In contrast, the maximum methyl-deuteron coupling for [1+/2H-SAM], which originates solely in the distance-dependent dipolar interaction, is somewhat smaller than in PFL-AE (A(2H)max ) 0.7 vs 1.0 MHz).23 The ENDOR results from four isotopic labels show that both enzymes exhibit the same motif for SAM binding to the [4Fe-4S]+ cluster of the two enzymes: chelation of the unique cluster iron by the amino acid moiety of the SAM methionine; close proximity of the methyl group to the cluster. However, there appear to be significant, and possibly mechanistically important, differences in the details of the binding geometry of SAM. ENDOR spectra from [2+/SAM]red prepared with each of the four labels, 17O, 15N (Figure 1), and 13C, 2H (not shown), are indistinguishable from the corresponding spectra from [1+/SAM], indicating that SAM binds with essentially the same geometry in [2+/SAM] and [1+/SAM]. Thus, the interactions of SAM with the cluster and the active-site pocket appear to be independent of the oxidation state of the cluster. Correlating the ENDOR results with the earlier selenium XAS data obtained with SeSAM,22 we propose the mechanism in Scheme 1 for the reversible cleavage of SAM at the active site of LAM. Cleavage begins with SAM bound to the unique Fe of the [4Fe-4S] cluster by the carboxylato and amino ligands, with the sulfonium group held close to the cluster. Electron transfer then homolytically cleaves the C5′-S bond of SAM to form the 5′deoxyadenosyl radical, while the sulfur of methionine becomes the sixth ligand to the unique iron. Such a six-coordinated octahedral geometry for the unique Fe of a [4Fe-4S] cluster is well documented for aconitase.29,30 This mechanism allows for reversible cleavage of SAM, as required in the LAM mechanism. In an alternative cleavage mechanism put forward for PFL-AE, the sulfur becomes bonded to a sulfide in the cluster.24 In contrast to LAM, the cleavage of SAM by PFL-AE is irreversible. Acknowledgment. D.C. and P.A.F. gratefully acknowledge the contributions to this work of Professor George H. Reed, who contributed the H217O and encouraged us in this work, and of Dr. Olafur Th. Magnusson, who assisted in the enzymatic preparation of 13C-SAM. C.W. and B.M.H. are indebted to Dr. R. Davydov for sample irradiation and his cryoreduction expertise. This work has been supported by the NIH (Grants DK28607, P.A.F.; HL13531, B.M.H.). Supporting Information Available: EPR spectra of [2+/AdoMet]red and [1+/AdoMet] (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.
(9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25)
(26) (27)
(28) (29) (30)
Frey, P. A.; Magnusson, O. Th. Chem. ReV. 2003, 103, in press. Moss, M. L.; Frey, P. A. J. Biol. Chem. 1987, 262, 14859. Baraniak, J.; Moss, M. L.; Frey, P. A. J. Biol. Chem. 1989, 264, 1357. Ballinger, M. D.; Frey, P. A.; Reed, G. H. Biochemistry 1992, 31, 10782. Ballinger, M. D.; Frey, P. A.; Reed, G. H.; LoBrutto, R. Biochemistry 1995, 34, 10086. Chang, C. H.; Ballinger, M. D.; Reed, G. H.; Frey, P. A. Biochemistry 1996, 35, 11081. Wu, W.; Lieder, K. W.; Reed, G. H.; Frey, P. A. Biochemistry 1995, 34, 10532-10537. Wu, W.; Booker, S.; Lieder, K. W.; Bandarian, V.; Reed, G. H.; Frey, P. A. Biochemistry 2000, 39, 9561. Magnusson, O. Th.; Reed, G. H.; Frey, P. A. Biochemistry 2001, 40, 7773. Sofia, H. J.; Chen, G.; Hetzler, B. G.; Reyes-Spindola, J. F.; Miller, N. E. Nucleic Acids Res. 2001, 29, 1097. Petrovich, R. M.; Ruzicka, F. J.; Reed, G. H.; Frey, P. A. J. Biol. Chem. 1991, 266, 7656. Petrovich, R. M.; Ruzicka, F. J.; Reed, G. H.; Frey, P. A. Biochemistry 1992, 31, 10774. Lieder, K. W.; Booker, S.; Ruzicka, F. J.; Beinert, H.; Reed, G. H.; Frey, P. A. Biochemistry 1998, 37, 2578. Ruzicka, F. J.; Lieder, K. W.; Frey, P. A. J. Bacteriol. 2000, 182, 469476. Cheek, J.; Broderick, J. B. J. Biol. Inorg. Chem. 2001, 6, 209. Broderick, J. B.; Henshaw, T. F.; Cheek, J.; Wojtuszewski, K.; Smith, S. R.; Trojan, M. R.; McGhan, R. M.; Kopf, A.; Kibbey, M.; Broderick, W. E. Biochem. Biophys. Res. Commun. 2000, 269, 451. Rebeil, R.; Nicholson, W. L. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9038. Tamarit, J.; Mulliez, E.; Meier, C.; Trautwein, A.; Fontecave, M. J. Biol. Chem. 1999, 274, 31291. Sanyal, I.; Cohen, G.; Flint, D. H. Biochemistry 1994, 33, 3625. Me´jean, A.; Tse Sum Bui, B.; Florentin, D.; Ploux, O.; Izumi, Y.; Marquet, A. Biochem. Biophys. Res. Commun. 1995, 217, 1231. Ugulava, N. B.; Frederick, K. K.; Jarrett, J. T. Biochemistry 2003, 42, 2708-2719. Cosper, N. J.; Booker, S. J.; Frey, P. A.; Scott, R. A. Biochemistry 2000, 39, 15668. Cosper, N. J.; Booker, S. J.; Frey, P. A.; Scott, R. A. Biochemistry 2000, 39, 15668. Walsby, C.; Hong, W.; Broderick, W. E.; Cheek, J.; Ortillo, D.; Broderick, J. B.; Hoffman, B. J. Am. Chem. Soc. 2002, 124, 3143-3151. Walsby, C. J.; Ortillo, D.; Broderick, J. B.; Hoffman, B. J. Am. Chem. Soc. 2002, 124, 11270-11271. (a) L-[methyl-2H3]Methionine, L-[methyl-13C]methionine, and L-[2-15N]methionine were obtained from commercial suppliers. L-[1-17O]Methionine was prepared by acid-catalyzed exchange of L-methionine with H217O. Acid-catalyzed exchange of carboxyl oxygens with H218O has been described.26 Mass spectrometric analysis of the [1-17O]methionine indicated an 17O-enrichment of ∼48.5%. Taking into account the equivalence of carboxylate oxygens and the 16O, 17O, and 18O content of the H217O used, the isotopic profile of the exchanged methionine at C1 was as follows: 16O , 10.4%; 17O , 40%; 18O , 20%; 16O , 4.5%; 17O , 17%; 18O , 8.6%. 2 2 2 1 1 1 The labeled samples of L-methionine were used with ATP and SAM synthetase to produce labeled SAM as previously described, with minor modifications.23,24 35 GHz CW/pulsed EPR and pulsed ENDOR experiments were carried out at 2 K as previously described. Emptage, M. H.; Kent, T. A.; Kennedy, M. C.; Beinert, H.; Mu¨nck, E. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 4674-4678. ENDOR samples were prepared as follows inside an anaerobic chamber. The purified LAM was prepared as described.13,14 Samples (∼90 µM) were reductively incubated with 10 mM dihydrolipoate in the presence of 0.42 mM PLP, 1.0 mM ammonium iron (II) sulfate, and 15% glycerol in 42 mM Epps buffer at pH 8.0 and 37 °C for 4 h. The reductively incubated enzyme was concentrated by ultrafiltration through Microcon 30 (Millipore) to ∼500 µM. The concentrated enzyme was mixed with SAM or labeled SAM in the presence or absence of 6 mM sodium dithionite, transferred to ENDOR sample tubes, and quickly frozen in liquid nitrogen. Iron-sulfur clusters of samples prepared in the absence of dithionite were cryoreduced by γ-irradiation as desribed.23,24 In all samples, the concentrations of the enzyme and SAM were ∼400 µM and 3.7 mM, respectively. g1 is obscured by signals from a Mn2+ impurity, but examination of the field dependence of the ENDOR indicates that g1 ) 2.00-2.05. Werst, M. M.; Kennedy, M. C.; Beinert, H.; Hoffman, B. M. Biochemistry 1990, 29, 10526-10532. Kennedy, M. C.; Stout, C. D. AdV. Inorg. Chem. 1992, 38, 323-339.
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